人N-乙酰半乳糖胺-6-硫酸酯硫酸酯酶的纯化及性质

Purification and properties of human N-acetylgalactosamine-6-sulfate sulfatase.

作者信息

Lim C T, Horwitz A L

出版信息

Biochim Biophys Acta. 1981 Feb 13;657(2):344-55. doi: 10.1016/0005-2744(81)90320-x.

DOI:10.1016/0005-2744(81)90320-x

Abstract

Human N-acetylgalactosamine-6-sulfate sulfatase (EC 3.1.6.-) from human placenta has been purified more than 3000-fold by gel filtration, ion-exchange and substrate affinity chromatography. The enzyme has a molecular weight of 90 000 by gel filtration chromatography and 85 000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Enzyme purified from cultured human skin fibroblasts has similar properties. 2. The tritium-labeled chrondroitin 6-sulfate trisaccharide N-acetylgalactosamine 6-sulfate-(beta, 1-4)-glucuronic acid-(beta, 1-3(-N-acetyl[1-3H]galactosaminitol 6-sulfate as substrate demonstrated a Km of 0.12 mM at pH 4.5. Sulfate was hydrolyzed only from the non-reducing terminal of this disulfated trisaccharide. Hyaluronic acid, dermatan sulfate, chondroitin 4-sulfate, heparin and chondroitin 6-sulfate tetrasaccharide were slightly inhibitory, whereas 6-sulfated pentasaccharides and heptasaccharides were strongly inhibitory. The enzyme dose not hydrolyze sulfate from N-acetylglucosamine 6-sulfate.

摘要

人胎盘来源的人N-乙酰半乳糖胺-6-硫酸酯硫酸酯酶（EC 3.1.6.-）已通过凝胶过滤、离子交换和底物亲和色谱法纯化了3000多倍。通过凝胶过滤色谱法测定该酶的分子量为90000，通过十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳测定为85000。从培养的人皮肤成纤维细胞中纯化的酶具有相似的性质。2. 以氚标记的硫酸软骨素6-硫酸三糖N-乙酰半乳糖胺6-硫酸酯-（β，1-4）-葡萄糖醛酸-（β，1-3）-N-乙酰[1-³H]半乳糖胺醇6-硫酸酯作为底物，在pH 4.5时测得Km为0.12 mM。仅从这种二硫酸化三糖的非还原末端水解硫酸盐。透明质酸、硫酸皮肤素、硫酸软骨素4-硫酸酯、肝素和硫酸软骨素6-硫酸四糖有轻微抑制作用，而6-硫酸化五糖和七糖有强烈抑制作用。该酶不水解N-乙酰葡糖胺6-硫酸酯中的硫酸盐。